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Gut microbial colonization orchestrates TLR2 expression, signaling and epithelial proliferation in the small intestinal mucosa.

Hörmann N, Brandão I, Jäckel S, Ens N, Lillich M, Walter U, Reinhardt C - PLoS ONE (2014)

Bottom Line: The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment.TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2) and protein-kinase B (AKT) induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K.In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Junior Group Translational Research in Thrombosis and Hemostasis, Mainz, Germany.

ABSTRACT
The gut microbiota is an environmental factor that determines renewal of the intestinal epithelium and remodeling of the intestinal mucosa. At present, it is not resolved if components of the gut microbiota can augment innate immune sensing in the intestinal epithelium via the up-regulation of Toll-like receptors (TLRs). Here, we report that colonization of germ-free (GF) Swiss Webster mice with a complex gut microbiota augments expression of TLR2. The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment. TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2) and protein-kinase B (AKT) induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K. Mice that were colonized from birth with a normal gut microbiota (conventionally-raised; CONV-R) showed signs of increased small intestinal renewal and apoptosis compared with GF controls as indicated by elevated mRNA levels of the proliferation markers Ki67 and Cyclin D1, elevated transcripts of the apoptosis marker Caspase-3 and increased numbers of TUNEL-positive cells per intestinal villus structure. In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis. Our findings suggest that a tuned proliferation response of epithelial cells following microbial colonization could aid to protect the host from its microbial colonizers and increase intestinal surface area.

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Agonist-specific orchestration of the TLR expression profile in a mouse small intestinal epithelial cell line.a+b, Relative mRNA expression of TLR2 in MODE-K cells stimulated with PG (50 µg/ml) or Pam3CSK4 (0.5 µg/ml) for 2, 4 or, 8 hours (n = 4). c, TLR2 immunoblot of PG or Pam3CSK4 stimulated MODE-K cells. Cells were treated with or without (CTR) PG or Pam3CSK4 for 2, 4, or 8 hours (n = 3, representative blot). d–f, Relative TLR1, 6 and 4 mRNA expression in MODE-K cells stimulated with Pam3CSK4 or LPS for 2, 4, or 8 hours (n = 4). Results are shown as means ± s.e.m. One asterisk, P<0.05; two asterisks, P<0.01; three asterisks, P<0.005; four asterisks, P<0.001.
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pone-0113080-g003: Agonist-specific orchestration of the TLR expression profile in a mouse small intestinal epithelial cell line.a+b, Relative mRNA expression of TLR2 in MODE-K cells stimulated with PG (50 µg/ml) or Pam3CSK4 (0.5 µg/ml) for 2, 4 or, 8 hours (n = 4). c, TLR2 immunoblot of PG or Pam3CSK4 stimulated MODE-K cells. Cells were treated with or without (CTR) PG or Pam3CSK4 for 2, 4, or 8 hours (n = 3, representative blot). d–f, Relative TLR1, 6 and 4 mRNA expression in MODE-K cells stimulated with Pam3CSK4 or LPS for 2, 4, or 8 hours (n = 4). Results are shown as means ± s.e.m. One asterisk, P<0.05; two asterisks, P<0.01; three asterisks, P<0.005; four asterisks, P<0.001.

Mentions: To further pinpoint the underlying cellular mechanism of these microbial effects on TLR2 and TLR4 expression we used a sterile infection cell culture model. The small intestinal epithelial cell line MODE-K [19] was stimulated for 2, 4 and 8 hours with the bacterial TLR2-agonists peptidoglycan (PG), the synthetic lipoprotein TLR2-agonist Pam3CSK4 and the TLR4-agonist lipopolysaccharide (LPS). TLR2 transcript levels were strongly increased 2 and 4 hours after PG challenge (Fig. 3a) and TLR2 transcripts were also enhanced after Pam3CSK4 stimulation (Fig. 3b). Interestingly, these effects were blunted 8 hours after stimulation with PAMPs. This robust increase in TLR2 expression was also found on the protein level when MODE-K cells were stimulated with PG or Pam3CSK4 (Fig. 3c). Transcripts of the TLR2 co-receptor TLR1 were only slightly up-regulated upon longer stimulation periods with the TLR2 agonist Pam3CSK4 (Fig. 3d), while TLR6 expression was diminished at longer stimulation periods (Fig. 3e). In contrast to TLR4 expression in the small intestine of colonized mice (Fig. 1e) and to antibiotic decimation of the gut microbiota (Fig. 1j), treatment with LPS for 4h resulted in decreased TLR4 mRNA levels in MODE-K cells (Fig. 3f). Collectively, these results demonstrate an agonist-specific orchestration of the TLR expression profile in small intestinal epithelial cells.


Gut microbial colonization orchestrates TLR2 expression, signaling and epithelial proliferation in the small intestinal mucosa.

Hörmann N, Brandão I, Jäckel S, Ens N, Lillich M, Walter U, Reinhardt C - PLoS ONE (2014)

Agonist-specific orchestration of the TLR expression profile in a mouse small intestinal epithelial cell line.a+b, Relative mRNA expression of TLR2 in MODE-K cells stimulated with PG (50 µg/ml) or Pam3CSK4 (0.5 µg/ml) for 2, 4 or, 8 hours (n = 4). c, TLR2 immunoblot of PG or Pam3CSK4 stimulated MODE-K cells. Cells were treated with or without (CTR) PG or Pam3CSK4 for 2, 4, or 8 hours (n = 3, representative blot). d–f, Relative TLR1, 6 and 4 mRNA expression in MODE-K cells stimulated with Pam3CSK4 or LPS for 2, 4, or 8 hours (n = 4). Results are shown as means ± s.e.m. One asterisk, P<0.05; two asterisks, P<0.01; three asterisks, P<0.005; four asterisks, P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone-0113080-g003: Agonist-specific orchestration of the TLR expression profile in a mouse small intestinal epithelial cell line.a+b, Relative mRNA expression of TLR2 in MODE-K cells stimulated with PG (50 µg/ml) or Pam3CSK4 (0.5 µg/ml) for 2, 4 or, 8 hours (n = 4). c, TLR2 immunoblot of PG or Pam3CSK4 stimulated MODE-K cells. Cells were treated with or without (CTR) PG or Pam3CSK4 for 2, 4, or 8 hours (n = 3, representative blot). d–f, Relative TLR1, 6 and 4 mRNA expression in MODE-K cells stimulated with Pam3CSK4 or LPS for 2, 4, or 8 hours (n = 4). Results are shown as means ± s.e.m. One asterisk, P<0.05; two asterisks, P<0.01; three asterisks, P<0.005; four asterisks, P<0.001.
Mentions: To further pinpoint the underlying cellular mechanism of these microbial effects on TLR2 and TLR4 expression we used a sterile infection cell culture model. The small intestinal epithelial cell line MODE-K [19] was stimulated for 2, 4 and 8 hours with the bacterial TLR2-agonists peptidoglycan (PG), the synthetic lipoprotein TLR2-agonist Pam3CSK4 and the TLR4-agonist lipopolysaccharide (LPS). TLR2 transcript levels were strongly increased 2 and 4 hours after PG challenge (Fig. 3a) and TLR2 transcripts were also enhanced after Pam3CSK4 stimulation (Fig. 3b). Interestingly, these effects were blunted 8 hours after stimulation with PAMPs. This robust increase in TLR2 expression was also found on the protein level when MODE-K cells were stimulated with PG or Pam3CSK4 (Fig. 3c). Transcripts of the TLR2 co-receptor TLR1 were only slightly up-regulated upon longer stimulation periods with the TLR2 agonist Pam3CSK4 (Fig. 3d), while TLR6 expression was diminished at longer stimulation periods (Fig. 3e). In contrast to TLR4 expression in the small intestine of colonized mice (Fig. 1e) and to antibiotic decimation of the gut microbiota (Fig. 1j), treatment with LPS for 4h resulted in decreased TLR4 mRNA levels in MODE-K cells (Fig. 3f). Collectively, these results demonstrate an agonist-specific orchestration of the TLR expression profile in small intestinal epithelial cells.

Bottom Line: The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment.TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2) and protein-kinase B (AKT) induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K.In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Junior Group Translational Research in Thrombosis and Hemostasis, Mainz, Germany.

ABSTRACT
The gut microbiota is an environmental factor that determines renewal of the intestinal epithelium and remodeling of the intestinal mucosa. At present, it is not resolved if components of the gut microbiota can augment innate immune sensing in the intestinal epithelium via the up-regulation of Toll-like receptors (TLRs). Here, we report that colonization of germ-free (GF) Swiss Webster mice with a complex gut microbiota augments expression of TLR2. The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment. TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2) and protein-kinase B (AKT) induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K. Mice that were colonized from birth with a normal gut microbiota (conventionally-raised; CONV-R) showed signs of increased small intestinal renewal and apoptosis compared with GF controls as indicated by elevated mRNA levels of the proliferation markers Ki67 and Cyclin D1, elevated transcripts of the apoptosis marker Caspase-3 and increased numbers of TUNEL-positive cells per intestinal villus structure. In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis. Our findings suggest that a tuned proliferation response of epithelial cells following microbial colonization could aid to protect the host from its microbial colonizers and increase intestinal surface area.

Show MeSH
Related in: MedlinePlus